Rational drug development is a process of developing lead molecules, not by randomly screening thousands of molecules in the blind hope of finding one that shows the desired activity, but rather by deducing the active site of the target and devising a chemical that interacts with that site in the appropriate manner.
Epidermal Growth Factor Receptor (EGFR) is a member of the ErbB (HER) family receptor tyrosine kinase (RTKs), which regulate cell growth and differentiation and are implicated in many human cancers. EGFR activation and dimerization is discussed in, for example, Burgess et al. (2003) Molecular Cell 12, 541-552 and Ferguson et al. (2003) Molecular Cell 11, 507-517.
EGF activates its receptor by inducing dimerization of the extracellular region of EGFR. The activation of EGFR has been described through results of disulfide bond mapping as well as X-ray crystal structures. The crystal structures of ligand-bound sEGFR showed that dimerization is receptor mediated, with two individual ligand molecules present in the dimer. The dimerization interface of activated EGFR is completely occluded by intramolecular interactions, and is an autoinhibited configuration. To activate the receptor, a large domain rearrangement that exposes this occluded interface must accompany EGF binding where EGF does not contribute to the EGFR dimer interface. The EGFR mechanism is in sharp contrast to most other receptor tyrosine kinase activation mechanisms in which the bound ligand contributes directly to the receptor dimerization interface and does not dramatically alter the conformation of the extracellular region of the receptor tyrosine kinase.
EGFR contains four subdomain I, II, III, and IV. Almost all receptor/receptor contacts observed in the crystal structures of EGFR are mediated by domain II. At the center of the dimer interface is a prominent loop (residues 242-259 of EGFR) that extends from the second C1 module (module 5) of each domain II and reaches across the interface to interact primarily with domain II of its dimerization partner. This domain II loop, which is specific to ErbB receptors, is the “dimerization arm”. The dimerization arm of domain II is completely occluded by intramolecular interactions with domain IV (i.e., an autoinhibited configuration). There are two smaller interaction sites in the dimer that involve side chains from the second and the sixth disulfide-bonded modules of domain II. And the dimer interface may extend into domain IV. While the two receptor molecules approach one another very closely toward the C terminus of domain IV, a well-defined, tight interface is not observed.
Although EGF and TGF-α clearly do not span the dimer interface, each ligand simultaneously contacts two separate binding surfaces in the same EGFR molecule. The bound EGF or TGF-α molecule resembles a wedge between domains I and III. The relationship between domains I and II is essentially identical to that seen in IGF-1R and in the activated sEGFR dimer, implying that ligand binding does not greatly influence the relative orientation of these two domains. But the relationship between domains II and III differs dramatically in the activated and unactivated structures. A direct intramolecular interaction between cysteine-rich domains II and IV restrains the domain II/III relationship that characterizes the unactivated configuration. This interdomain “tether” is stabilized by essentially identical interactions between the two cysteine rich domains (II and IV) in inactive sErbB3 and sEGFR.
The intramolecular domain II/IV tether precisely buries the dimerization arm of domain II against domain IV, so that the tethered configurations of sErbB3 and sEGFR cannot dimerize and thus appear to be autoinhibited. Moreover, the two ligand binding surfaces on domain I and III are too far apart in the tethered configuration for a single ligand to bind to both simultaneously. Consequently, the tethered configuration can only form low-affinity interactions with ligand, using just one of its ligand binding surfaces at a time.
Switching between the unactivated and activated configurations of sEGFR requires domains I and III to be drawn toward one another through a 130° rotation of the rigid domain I/II pair in one plane and a 20° rotation in another. Only this extended configuration of sEGFR is capable of both high-affinity ligand binding and efficient dimerization.
Based upon energetic calculations, it is currently thought that at any given time, about 95% of sEGFR molecules will be tethered and the remaining 5% will not. The presence of ligand and subsequent binding to domains I and III of the non-tethered form will drive the equilibrium toward the non-tethered form, trapping receptor molecules in the extended state that can dimerize.
Exposure of the dimerization arm is not sufficient alone to drive EGFR dimerization. Also required is additional contact sites in modules 2 and 6 of domain III. These two additional contact sites and the dimerization arm cooperate at the dimer interface.
Known strategies of EGFR inhibition are directed to antibody binding of domain III to provide steric hindrance of the required configuration change (e.g., Erbitux). Other conventional strategies are directed to antibody binding of domain II, specifically the dimerization arm, so as to prevent dimerization (e.g., pertuzumab). Still other conventional strategies are directed to antibody binding of domain IV residues that participate in the intramolecular tether (e.g., trastuzumab, Herceptin). But no existing strategies are directed to the tethering mechanism of activation.